Proteinase activities in quiescent and germinating seeds of xHaynaldoticum sardoum

Breakdown of gliadin during germination of xHaynaldoticum sardoum Meletti et Onnis seeds is correlated with the appearance in the endosperms of a proteinase activity, which is absent in the quiescent seed. This activity is optimal at pH 4 and has a maximum stability at pH 4–5. Gel filtration of proteinase activity extracted from quiescent seeds indicates a molecular weight of 60–100 kDa. The proteinase can hydrolyze hemoglobin but not gliadin and is inhibited by pepstatin A and, to a lower extent, by p -chloromercuribenzoic acid (p-CMB). Gel filtrations of crude extracts from germinating seeds reveal two peaks (molecular weight 66 and 21 kDa) of activity against hemoglobin and a shoulder and a peak (molecular weight 21 kDa) of activity on gliadin. The first peak of activity against hemoglobin is inhibited by pepstatin A and p-CMB; the second one is inhibited by p-CMB and leupeptin. As for the gliadin-eluted activity the shoulder is mainly inhibited by pepstatin A and p-CMB, whereas the peak is inhibited by p-CMB and leupeptin. Estimations of the ratios of total nitrogen to α-amino nitrogen, suggest that the enzyme preparations mainly contain proteinases. It is concluded that the proteinases present in the quiescent seeds of xH. sardoum , in particular aspartic proteinases (EC 3.4.23), could play a role as initiator endoproteases or participate in the digestion of modified proteins during the mobilization of reserve proteins. The cysteine proteinases (EC 3.4.22) appearing during the germination seem to account for the hydrolysis of the most abundant class of protein reserves, the prolamins.     

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.     

Interaction of pig kidney and lentil seedling copper-containing amine oxidases with guanidinium compounds

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Ki value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.     

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.     

Arginine and ornithine oxidation catalyzed by lentil seedling copper-amine oxidase

The oxidation of L-ornithine and L-arginine catalyzed by lentil (Lens esculenta) seedling copper-amine oxidase has been investigated by polarographic techniques, optical spectroscopy, and capillary electrophoresis. Both L-ornithine and L-arginine were found to be poor substrates for lentil amine oxidase. L-Ornithine was oxidized to glutamate-5-semialdehyde and ammonia, in similar manner as usual substrates. Glutamate-5-semialdehyde spontaneously cyclizes to ¹-pyrroline-5-carboxylic acid. Arginine is oxidized by an unusual mechanism yielding glutamate-5-semialdehyde, ammonia, and urea as reaction products.     

Reversible thermal inactivation and conformational states in denaturant guanidinium of a calcium-dependent peroxidase from Euphorbia characias

The changes in the heme environment and overall structure occurring during reversible thermal inactivation and in denaturant guanidinium of Euphorbia characias latex peroxidase (ELP) were investigated in the presence and absence of calcium ions. Native active enzyme had an absorption spectrum typical of a quantum-mixed spin ferric heme protein. After 40 min at 60 degrees C ELP was fully inactivated showing the spectroscopic behavior of a pure hexacoordinate low-spin protein. The addition of Ca2+ to the thermally inactivated enzyme restored its native activity and its spectroscopic features, but did not increase the stability of the protein in guanidinium. It is concluded that, in Euphorbia peroxidase, Ca2+ ion play a key role in conferring structural stability to the heme environment and in retaining active site geometry.     

Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN--gp120 interaction

One of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, of which the best studied example is DC-SIGN, which mediates HIV-1 internalization. DC can keep the virus infectious for several days and are able to transmit HIV-1 to CD4(+) T cells. We tested proteins from milk and serum for their ability to block DC-mediated HIV-1 transmission, of which bovine lactoferrin (bLF) is the most potent inhibitor. bLF binds strongly to DC-SIGN, thus preventing virus capture and subsequent transmission. Interestingly, bLF is a much more efficient inhibitor of transmission than human lactoferrin. Since bLF is nontoxic and easy to purify in large quantities, it is an interesting candidate microbicide against HIV-1. Another advantage of bLF is its ability to block HIV-1 replication in T cells. DC-mediated capture of a bLF-resistant HIV-1 variant that was selected during long-term culturing in T cells could still be blocked by bLF. This underscores the usefulness of bLF as a microbicide drug to prevent HIV-1 transmission.     

Coefficient of relationship by isonymy between 14 Sardinian villages in the periods 1800-1824 and 1950-1974

Lasker's coefficient of relationship was calculated between 14 villages in Sardinia in order to estimate biological relationships on the base of common surnames. The data derive from parish marriage registers for the periods 1800-1824 and 1950-1974. Through time, coefficients of relationship between villages are generally higher for neighbouring villages. Moreover, the Ri between values decreases as the geographic distances increase. The negative Pearson product-moment correlation observed between the Ri matrix and the geographic distance matrix is statistically significant (Mantel's test) for the two periods considered. These results suggest that the biological similarity between villages, as shown by isonymy, tends to decrease as the geographic distance increases. In addition, the plots of isonymic relationships obtained by nonmetric multidimensional scaling for 1800-1824 and 1950-1974 show that the biological relationships between neighbouring villages increase in the second period considered.     

Diamine oxidase from Lens esculenta seedlings: Purification and properties

A diamine oxidase (DAO) (EC 1.4.3.6) has been purified to homogeneity from lentil seedlings. The purified protein has a MW of 154 000 and is composed of two apparently identical subunits. It contains two CU²⁺ atoms and one carbonyl-like group per mol. The purified enzyme is pink-red in concentrated solution and shows a broad, well-defined, absorption band in the visible region centered at 498 nm. The ESR spectrum is typical of Cu²⁺ in a tetragonal symmetry. The enzyme oxidizes only aliphatic diamines and spermidine with formation of the corresponding aldehydes, hydrogen peroxide and ammonia. Putrescine and cadaverine are oxidized most rapidly and the oxidation rate decreases when longer diamines are tested.